Iterative SCRaMbLE for engineering synthetic genome modules and chromosomes.

Saccharomyces cerevisiae is closing-in on the first synthetic eukaryotic genome with genome-wide redesigns, including LoxPsym site insertions that enable inducible genomic rearrangements in vivo via Cre recombinase through SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxPsym-mediated Evolution). Combined with selection, SCRaMbLE quickly generates phenotype-enhanced strains by diversifying gene arrangement and content. Here, we demonstrate how iterative cycles of SCRaMbLE reorganises synthetic genome modules and chromosomes to improve functions. We introduce SCOUT (SCRaMbLE Continuous Output and Universal Tracker), a reporter system that allows sorting of SCRaMbLEd cells into high-diversity pools. Paired with long-read sequencing, SCOUT enables high-throughput mapping of genotype abundance and genotype-phenotype relationships. Iterative SCRaMbLE is applied here to yeast strains with a full synthetic chromosome and histidine biosynthesis modules. Five HIS module designs are tested, and SCRaMbLE is used to optimise the poorest performer. Our results highlight iterative SCRaMbLE as a powerful tool for data driven modular genome design.